Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.

Isolation of Clostridium difficile from fecal specimens has been facilitated by the development of a selective and differential medium, cefoxitin-cycloserinefructose agar (CCFA). We substituted 0.1% sodium taurocholate for the 2.5% egg yolk in CCFA and compared the growth of 15 isolates of C. difficile on the resulting medium with growth on conventional CCFA. The taurocholate-containing medium (TCCFA) quantitatively recovered vegetative forms of C. difficile in the same numbers as CCFA medium. Recovery of spores was a mean 1.7 log(10) higher on TCCFA than on CCFA. Thirty-six of 60 patient stool specimens growing C. difficile gave a heavier growth on TCCFA than on CCFA, and 9 failed to yield C. difficile on CCFA. TCCFA detected spores of 75 colony-forming units per ml from artificially inoculated fecal specimens when conventional stool culturing techniques were used. Fluorescence of colonies of C. difficile was more intense on TCCFA than on CCFA. TCCFA was simpler to prepare and, overall, was more sensitive than CCFA.     

Selective and differential medium for isolation of Clostridium difficile.

Clostridium difficile is a recognized cause of pseudomembranous (antimicrobial agent-associated) colitis and may be one of the causes of antimicrobial agent-induced diarrhea. A selective and differential agar medium that contains cycloserine, cefoxitin, fructose, and egg yolk (CCFA) was developed to facilitate the isolation of C. difficile from fecal specimens. Quantitative cultures of 16 stock strains of C. difficile on this medium (and on a medium containing cycloserine, fructose, and egg yolk) yielded counts equivalent to those obtained on blood agar; other media selective for clostridia, including Clostrisel agar, reinforced clostridial agar plus 0.2% para-cresol, and egg yolk-neomycin agar (the latter was inoculated with cultures subjected to prior heat shocking), were also tested and found to be inhibitory to the growth of C. difficile. Of 28 fecal or colostomy effluent specimens cultured on the above media, 14 yielded C. difficile. CCFA was found to be the most sensitive and selective of these media for the recovery of C. difficile. Colonies of C. difficile growing on CCFA had distinctive morphological and fluorescent properties which were sufficient for presumptive identification. CCFA should provide a rapid method for the screening of fecal specimens from patients with antimicrobial agent-associated diarrhea or colitis for C. difficile.     

New selective medium for isolating Clostridium difficile from faeces.

AIMS: To compare CCFA (cycloserine, cefoxitin fructose agar) with a new selective medium CDMN (containing cysteine hydrochloride, norfloxacin, and moxalactam) for the isolation of Clostridium difficile after direct faecal culture. METHODS: The minimum inhibitory concentration (MIC) of norfloxacin was determined for 64 strains of C difficile, 17 strains of other Clostridium sp, and 66 various isolates of faecal origin, together with MIC determinations of moxalactam against the 81 strains of Clostridium sp and 15 isolates of Bacteroides sp. Using C difficile agar base with 0.5 g/l of cysteine hydrochloride, norfloxacin and moxalactam were incorporated into the medium and compared with CCFA for the isolation of C difficile after direct faecal culture. RESULTS: Norfloxacin (12 mg/l) inhibited the growth of enterobacteriaceae and faecal streptococci; moxalactam (32 mg/l) inhibited the growth of most strains of Bacteroides sp tested, together with Clostridium sp other than C difficile. Using the antibiotics in combination (CDMN), the growth and colonial morphology of 64 strains of C difficile were unaffected. When CDMN medium was compared with CCFA for the isolation of C difficile from 832 faeces from inpatients with diarrhoea, the CDMN agar isolated 20% more strains and reduced the number of contaminating colonies by 30%. CONCLUSIONS: CDMN both improves the isolation rate of C difficile from faecal specimens and reduces the growth of other organisms compared with CCFA.     

Comparison of four commercial brucella agar media for growth of anaerobic organisms.

Four different commercial brucella blood agar plating media (Anaerobe Systems, BBL Microbiology Systems, Remel, and Scott Laboratories) were compared for the abilities to recover anaerobic organisms from clinical specimens and to support the growth of American Type Culture Collection anaerobic stock cultures. Following 24 h of incubation in an anaerobe chamber, Anaerobe Systems prereduced, anaerobically sterilized brucella plates yielded 63% of the total clinical anaerobe isolates, the Scott medium yielded 51%, the Remel medium yielded 42%, and the BBL medium yielded 37%. Poor growth of Peptostreptococcus magnus, P. anaerobius, Fusobacterium necrophorum, F. nucleatum, and pigmented Bacteroides spp. was observed on brucella media obtained from BBL, Remel, and Scott. Data obtained with stock anaerobic cultures showed that Anaerobe Systems plates yielded good growth and produced a larger colony size with all of the strains tested in 1 day, whereas poor growth of Peptostreptococcus spp., B. melaninogenicus, and Fusobacterium spp. was noted on brucella media from BBL, Remel, and Scott.     

Evaluation of Cycloserine-Cefoxitin Fructose Agar (CCFA), CCFA with Horse Blood and Taurocholate,and Cycloserine-Cefoxitin Mannitol Broth with Taurocholate and Lysozyme for Recovery of Clostridium difficile Isolates from Fecal Samples

Cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate (CCFA-HT), and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared for recovery of Clostridium difficile from 120 stool specimens. Compared to CCFA, CCFA-HT enhanced C. difficile growth and improved recovery by 4%. In a separate study, 9% (8/91) of stool samples previously C. difficile negative on plate medium were C. difficile positive when cultured in CCMB-TAL.     

Evaluation of a New Chromogenic Medium for the Isolation and Presumptive Identification of Salmonella Species from Stool Specimens

The performance of BBL CHROMagar Salmonella (Becton Dickinson, France), a new selective chromogenic medium for the isolation and presumptive identification of Salmonella spp., was evaluated. On this medium, which is a modification of CHROMagar Salmonella (CHROMagar Microbiology, France) with enhanced selectivity, the colonies of Salmonella are stained in mauve (rose-violet), while those of other organisms appear in blue-green or are not stained by any of the chromogens of the medium. The medium was evaluated with a total of 176 strains of Salmonella and other organisms, consisting of 18 reference strains and 158 clinical isolates. All Salmonella strains except subspecies IIIa and IIIb strains and Salmonella Gallinarum yielded typical mauve colonies. During the evaluation with 107 known positive and 332 unknown stool specimens in a clinical laboratory, a total of 115 and 105 Salmonella isolates were obtained on BBL CHROMagar Salmonella and Hektoen enteric agar, respectively. From the known positive stool specimens, 92 true positive cultures were obtained on BBL CHROMagar Salmonella and 89 on Hektoen enteric agar, yielding sensitivities of 86 and 83%, respectively. From the unknown stool specimens, a total of 27 Salmonella isolates were obtained, with 23 isolated from BBL CHROMagar Salmonella and 16 from Hektoen enteric agar by direct plating (sensitivity 85 and 59%, specificity 99 and 97%, respectively). Seroagglutination tests could be performed directly from BBL CHROMagar Salmonella. Compared to conventional isolation media, the time needed for confirmatory biochemical and serological tests was shortened by about 1 day when BBL CHROMagar Salmonella was used. On the basis of these results, the medium can be recommended for the primary isolation and presumptive identification of Salmonella spp. from clinical stool specimens. Electronic Publication     

"Second look" at cytotoxin B of Clostridium difficile in the course of diarrhea associated with antibiotic therapy]

Clostridium difficile is a sporulated obligate anaerobe responsible for most cases of antibiotic-associated colitis, for 15 to 25% of cases of antibiotic-related diarrhea, and for a substantial proportion of nosocomial infections. The most important laboratory test for the diagnosis of C. difficile infection is examination of the stool for C. difficile toxins A and/or B. Detection of cytotoxin B using the direct cytotoxicity assay (D-CA) is the gold standard test. Whether routine isolation of the organism from stool is warranted remains controversial. OBJECTIVES: To evaluate second-look CA done on C. difficile culture-positive filtrates from stool samples negative by the D-CA. METHODS: 300 consecutive stool samples sent to the Alfred Fournier Institute from April through October 1998 for a CA were routinely cultured on modified Cefoxitin Cycloserine Fructose Agar medium (CCFA). All CA-negative samples that grew C. difficile were examined by second-look CA. RESULTS: 245 stool specimens (81.7%) were negative by both CA and culture. The remaining 55 specimens all yielded C. difficile by culture; 32 (58.2%) had a positive D-CA and nine (16.4%) a negative D-CA with a positive second-look CA done on culture filtrates. CONCLUSION: Our data suggest that stool specimens sent for a direct CA should be routinely cultured to provide material for a second-look CA on culture-positive filtrates if the first CA prove negative. Culturing also allows to study antimicrobial drug resistance phenotypes and epidemiological markers.     

Comparison of Rambach agar, SM-ID medium, and Hektoen Enteric agar for primary isolation of non-typhi salmonellae from stool samples.

Stool samples (n = 504) were streaked simultaneously onto Rambach agar (R agar; E. Merck, Darmstadt, Germany), SM-ID medium (bioMérieux S.A., Montalieu-Vercieu, France), and Hektoen Enteric (HE) agar (BBL Becton-Dickinson, Baltimore, Md.) in order to evaluate the performances of the first two media in comparison with that of the well-established HE agar. Following overnight cultivation at 37 degrees C, 29 samples (5.8%) were positive for non-typhi salmonellae on at least one of the three media. Sensitivities and specificities were 69 and 98%, 79 and 85%, and 100 and 79% for R, SM-ID, and HE agars, respectively. On the basis of the poor sensitivities, R and SM-ID agars are not recommended as primary plating media when screening for non-typhi salmonellae. However, the high specificity of R agar may help to reduce the work load when this medium is used for plating after enrichment.     

Comparison of methods for recovery of Clostridium difficile from an environmental surface

Survival of Clostridium difficile in an aerobic environment is possible because of spore formation. When sodium taurocholate is substituted for the egg yolk of a selective medium, cycloserine-cefoxitin-fructose-agar (CCFA), enhanced recovery of C. difficile spores is shown. This selective medium (TCCFA) does not improve recovery of vegetative forms. In this study, dry and saline-moistened swabs, adhesive paddles, and Rodac plates containing CCFA and TCCFA were compared in their ability to recover C. difficile spores from an inoculated surface. Rodac plates grew 20 to 25 times as many spores on TCCFA as on CCFA. Saline-moistened swabs recovered fewer organisms than Rodac plates. Dry swabs and adhesive paddles rarely recovered spores. Prereduction of agar in an anaerobic chamber was not necessary for optimal spore recovery. Optimal growth of vegetative C. difficile required prereduced media. Agar prereduced for 2 h supported the growth of 12 C. difficile isolates as well as agar prereduced for 18 h. Vegetative cells of C. difficile survived for only 15 min in room air. Use of Rodac plates containing TCCFA is preferred for detection of C. difficile spores in the hospital environment.     

Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions

The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.     

Comparison of performance of the novel chromogenic spectra VRE agar to that of bile esculin azide and Campylobacter agars for detection of vancomycin-resistant enterococci in fecal samples

A total of 142 stool specimens were evaluated for vancomycin-resistant enterococcus (VRE). Twenty-four-hour sensitivities and specificities, respectively, were 98% and 95% for Spectra VRE chromogenic agar (Remel, Lenexa, KS), 86% and 92% for bile esculin azide with vancomycin (BEAV; Remel), and 96.5% and 92% for Campylobacter agar (CAMPY; Remel). Spectra VRE and CAMPY are significantly more sensitive at 24 h than BEAV.     

Evaluation of five new plating media for isolation of Salmonella species.

A three-phase study was conducted to compare Hektoen enteric agar (HE), Rambach agar (Ra), SM-ID medium (SM), xylose-lysine-Tergitol 4 agar (XLT4), novobiocin-brilliant green-glycerol-lactose agar (NBGL), and modified semisolid Rappaport-Vassiliadis medium (MSRV) for the recovery of nontyphoid salmonellae from stool specimens. After evaluation of the first two phases, which resulted in the elimination of Ra, SM, and NBGL, 593 consecutive stool samples were investigated by plating them directly and after tetrathionate enrichment at 37 degrees C on HE, XLT4, and MSRV. A total of 82 Salmonella-positive stool specimens were detected (positivity rate, 13.8%). Sensitivities for direct plating and after tetrathionate enrichment were 32.9 and 86.6%, respectively, for XLT4, 63.4 and 100.0%, respectively, for MSRV, and 34.1 and 79.3%, respectively, for HE. Specificities (percentage of morphologically suspicious colonies that were indeed salmonellae) were 100.0 and 99.8%, respectively, for XLT4, 99.0 and 98.8%, respectively, for MSRV, and 67.9 and 75.0%, respectively, for HE. The use of MSRV instead of HE increased the isolation rate of salmonellae by 26.2% (65 versus 82 strains isolated from HE and MSRV, respectively). We conclude that MSRV is the most sensitive medium tested and is a very specific medium for the isolation of nontyphoid salmonellae from stool specimens. However, its semisolid nature is a disadvantage and requires careful handling in the laboratory, especially when salmonellae are present. XLT4 had a sensitivity comparable to that of HE and a nearly 100% specificity and can be regarded as an alternative for the isolation of nontyphoid salmonellae from stool samples.     

Comparison of media for screening of diarrheic stools for the recovery of Clostridium difficile.

Recoveries of Clostridium difficile from stool specimens by using three media, cycloserine-mannitol agar (M-CMA), cycloserine-mannitol-blood agar (M-CMBA), and cycloserine-cefoxitin agar (M-CCA), were compared. Of 321 clinical specimens, 37 yielded C. difficile. Thirty-four were positive on M-CCA, 21 were positive on M-CMA, and 20 were positive on M-CMBA. M-CCA recovered significantly more C. difficile than did M-CMBA or M-CMA.     

Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile.

The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA). Fifty stored isolates of C. difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C. difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive. Other Clostridium species that were PRO Disc positive could be differentiated from C. difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining.     

Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin.

Stools from patients with antibiotic-associated diarrhea or colitis were cultured to detect the presence of Clostridium difficile. All specimens contained a cytotoxin which was neutralized by Clostridium sordellii antitoxin. Initial testing employed several methods with comparative merits in recovering this organism. These included the use of nonselective media, antibiotic-incorporated media, alcohol shock, and paracresol-containing broth. Optimal results were achieved with primary plating of serial dilutions onto a selective agar containing cycloserine and cefoxitin. This technique was then employed in a large number of specimens. The overall results showed that C. difficile was recovered in specimens from 71 of 73 patients. All isolates of C. difficile produced a cytotoxin which was neutralized by C. sordellii antitoxin in vitro. These results verify the utility of this medium and support the concept that C. difficile accounts for the cytotoxin found in stools in nearly all cases.     

Comparison of axenic and monoxenic media for isolation of Acanthamoeba.

Acanthamoeba is a genus of ubiquitous, free-living amebae that can be difficult to isolate by standard microbiologic techniques. We retrospectively reviewed the laboratory records of patients with ocular acanthamoebic infection for the period from January 1973 to June 1996 and found that Acanthamoeba isolates were recovered from 73, 71, and 70% of clinical specimens inoculated onto buffered charcoal-yeast extract agar (BCYE), nonnutrient agar with live or dead Escherichia coli, and tryptic soy agar (TSA) with horse or sheep blood, respectively. We then prospectively compared the recovery of a corneal isolate of Acanthamoeba on commercial media from Remel and BBL (TSA with 5% sheep blood, TSA with 5% horse blood, TSA with 5% rabbit blood, V agar, chocolate agar, BCYE, and selective BCYE with polymyxin B, anisomycin, and vancomycin) and on axenic and monoxenic media prepared with live or dead bacteria (Enterobacter aerogenes, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, and Stenotrophomonas maltophilia). Good recovery of trophozoites was obtained on BCYE, TSA with rabbit blood, TSA with horse blood, and Remel TSA with sheep blood. BBL TSA with horse blood or rabbit blood provided good recovery of cysts. All species of live or dead bacteria yielded good recovery of trophozoites; however, only nonnutrient agar with live P. aeruginosa, live E. aerogenes, or live S. maltophilia gave good recovery of cysts. TSA with either rabbit blood or horse blood, BCYE, and nonnutrient agar prepared with live P. aeruginosa, E. aerogenes, or S. maltophilia offer optimal recovery of Acanthamoeba.     

Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium difficile from environmental sites.

Cycloserine-cefoxitin-fructose agar (CCFA) and cycloserine-cefoxitin-fructose broth (CCFB) containing either 500 or 250 micrograms of cycloserine per ml were compared for efficacy in the isolation of Clostridium difficile from hospital ward environmental sites. A RODAC imprint technique was used to inoculate prereduced CCFA. Moistened swabs were used to inoculate prereduced CCFB from environmental sites immediately adjacent to the RODAC sample sites. CCFA (6% positive) was significantly more sensitive than CCFB (3% positive; P less than 0.005), regardless of the cycloserine concentration. When the CCFA cycloserine concentration was decreased from 500 to 250 micrograms/ml, the overall rate of positive cultures rose from 4 to 17%. Medium containing 500 micrograms of cycloserine per ml may be too inhibitory to isolate many moderately sensitive strains of C. difficile from environmental sites. Regardless of the cycloserine concentration, the CCFA RODAC imprint technique is superior to the CCFB method.     

Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori.

We developed a new agar, egg yolk emulsion (EYE) agar, for cultivation of Helicobacter pylori. EYE agar contains Columbia agar base (Oxoid), 10% EYE (Oxoid), 1% IsoVitaleX (BBL), and 40 mg of Triphenyleteraxolium chloride (Sigma) per liter. We compared EYE agar with the following agars: (i) brain heart infusion agar-7% horse blood-1% IsoVitaleX (GDW agar; C. S. Goodwin, E. D. Blincow, J. R. Warren, T. E. Waters, C. R. Sanderson, and L. Easton, J. Clin. Pathol. 38:1127-1131, 1985), (ii) brain heart infusion agar-10% horse serum-0.2% charcoal-1% yeast extract-40 mg of triphenyltetrazolium chloride per liter (GLU agar; Y. Glupczynski, M. Labbe, and F. Thiabaumont, p. 3-6, in F. Megraud and H. Lamouliatte, ed., Gastroduodenal Pathology and Campylobacter pylori, 1989), (iii) Columbia agar with 7% lysed horse blood (D&M agar; J. C. Dent and C. A. M. McNulty, Eur. J. Clin. Microbiol. Infect. Dis. 7:555-558, 1988), and (iv) brain heart infusion agar-10% EYE-1% IsoVitaleX (BHIE agar). H. pylori CFU counts, expressed as average percentages of maximum growth, were as follows: EYE agar, 96; GDW agar. 76; BHIE agar, 57; D&M agar, 52; and GLU agar, 23. Colony counts for EYE agar were significantly higher than for GDW agar (P = 0.027), BHIE agar (P = 0.005), D&M agar (P = 0.0001), and GLU agar (P less than 0.0001). EYE agar also had higher CFU counts than two commercial chocolate media; the EYE agar count was 80%, versus 33% for BBL chocolate medium and 63% for Remel chocolate medium.     

Evaluation of the Chromogenic Agar chromID C. difficile

Three selective media (chromID C. difficile agar, taurocholate cycloserine cefoxitin agar [TCCA; homemade], and CLO medium) were compared from 406 stool samples of patients suspected of having Clostridium difficile infection. The sensitivities of chromID C. difficile agar at 24 h and 48 h, CLO medium, and TCCA were 74.1%, 87%, 85.2%, and 70.4%, respectively.     

Comparison of Salmonella chromogenic medium with DCLS agar for isolation of Salmonella species from stool specimens

Salmonella chromogenic medium (SCM; Oxoid, Basingstoke, United Kingdom), a new selective chromogenic medium, was compared to DCLS agar (Oxoid) for the detection and presumptive identification of Salmonella species from stool samples. This medium contains two chromogenic substrates, Magenta-cap (5-bromo-6-chloro-3-indolylcaprylate), which is hydrolyzed by Salmonella species to give magenta colonies, and X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), which is incorporated to visualize beta-D-galactosidase-producing organisms as blue colonies. Thus, non-Salmonella organisms appear blue or are not stained by any of the chromogens of the medium. A total of 500 stool samples were investigated by plating them directly and after selenite enrichment on DCLS agar and SCM. A total of 44 Salmonella-positive stool samples were detected. The sensitivities for direct plating and after enrichment were 22.7 and 81.8%, respectively, for DCLS agar, and for SCM these values were 34.1 and 100%, respectively. The specificities for direct plating and after enrichment were 82.5 and 72.8%, respectively, for DCLS agar and 98.5 and 95.8%, respectively, for SCM. According to these results, the sensitivities of SCM and DCLS agar were comparable on primary plating. However, the sensitivity of SCM was significantly higher after enrichment. In addition, the specificity of SCM was also significantly higher than that of DCLS agar both before and after enrichment. On the basis of these results, SCM can be recommended for the isolation of Salmonella species from stool samples in preference to DCLS agar.